The activity of Levisticum officinale W.D.J. Koch essential oil against multidrug-resistant Mycobacterium tuberclosis.

Background and Objectives
Essential oils are used for controlling and preventing human diseases and the application of those can often be quite safe and effective with no side effect. The essential oils have been found to have antiparasitic, antifungal, antiviral, antioxidant and especially antibacterial activity including antibacterial activity against tuberculosis. In this study the chemical composition and anti-TB activity of essential oil extracted from Levisticum officinale has been evaluated.


Materials and Methods
The essential oil of L. officinale was obtained by the hydro distillation method and the oil was analyzed by GC-FID and GC-MS techniques. The antibacterial activity of essential oil was evaluated through Minimum Inhibitory Concentration (MIC) assay using micro broth dilution method against multidrug-resistant Maycobacterium tuberculosis. The molecular modeling of major compounds was evaluated through molecular docking using Auto Dock Vina against-2-trans-enoyl-ACP reductase (InhA) as key enzyme in M. tuberclosis cell wall biosynthesis.


Results
The hydrodistillation on aerial parts of L. officinale yielded 2.5% v/w of essential oil. The major compounds of essential oil were identified as α-terpinenyl acetate (52.85%), β- phellandrene (10.26%) and neocnidilide (10.12%). The essential oil showed relatively good anti-MDR M. tuberculosis with MIC = 252 μg/ml. The results of Molecular Docking showed that affinity of major compounds was comparable to isoniazid.


Conclusion
The essential oil of aerial parts extracted from L. officinale was relatively active against MDR M. tuberculosis, and molecular docking showed the major compounds had high affinity to inhibit 2-trans-enoyl-acyl carrier protein reductase (InhA) as an important enzyme in M. tuberculosis cell wall biosynthesis.


INTRODUCTION
Essential oils are beneficial for controlling and preventing human diseases and the application of those can often be quite safe and effective with no side effect. The essential oils have been found to have antifungal, antiparasitic, antiviral, antioxidant and especially antibacterial activity (1). The essential oil can be used for the treatment of different diseases including atherosclerosis, thrombosis and diabetic (2). Also the essential oils play an important role regarding inhibiting growth of M. tuberculosis and it can be mentioned that essential oils of Salvia aratocensis (Beijing genotype strains), Eugenia caryophyllata, Cuminum cyminum, and Cinnamomum verum (strain H37Rv) also have this feature as well (3)(4).
Levisticum officinale belongs to Apiaceae family and it grows in the Hezar Mountain located in Kerman province, Iran (5). L. officinale as a medicinal plant is used in the treatment of urinary tract infection and kidney stone (6). Its oil is used in medicinal preparation, food flavoring and aromatherapy (7).
Although the antibacterial activity of L. officinale extract has been evaluated on some Mycobacteri-0.25 µm film thickness) was used for GC-MS analysis. The temperature of the oven was set from 60 to 250ºC at 4ºC/min, and then isothermal for 15 min. The FID and injector temperatures were programmed at 240ºC and 250ºC, respectively. Helium was used as carrier gas at the constant flow rate of 1.1 ml/min and a split ratio of 1:100. Mass range was 45-450 amu and the MS operating parameters were: ion source temperature, 200ºC; ionization voltage, 70 eV (7).
GC-FID analysis. The Agilent gas chromatograph (FID) with a DB-5 fused silica column (30 m × 0.32 mm; 0.25 µm film thickness) was used to GC analysis. Nitrogen was used as a gas carrier at a constant flow of 1.1 mL/min, a split ratio of 1:50. The oven temperature was set from 60 to 250ºC at 4ºC/min, and then isothermal for 15 min. The injector and FID temperatures were programmed at 240ºC and 250ºC, respectively.
Identification of components. The constituents of the volatile oil were recognized by the calculation of their retention indices under temperature-proum species (8)(9), there is no report about anti-My-grammed conditions for n-alkanes (C -C ) and the 6 24 cobacterium activity of essential oil extracted from this plant. So in this study, we aimed at evaluating the antibacterial activity of L. officinale essential oil against MDR M. tuberculosis strain.

MATERIALS AND METHODS
Plant material. The aerial parts of L. officinale were collected in 2016, from the Hezar Mountain located in Kerman province, Iran. The plant material was identified by Prof. Farideh Attar. A voucher specimen (46553-TUH) has been deposited in the herbarium of Science Faculty of Tehran University.
Isolation of essential oil. The aerial parts (150 g) of L. officinale was crushed and then the essential oil was isolated by hydro distillation using a Clevenger type instrument for 3 hours. Finally, the oil was dried with anhydrous sodium sulfate and stored in dark vials at -20 o C before analysis.

Analysis of the essential oil: GC-MS analysis.
Thermoquest Finnigan Trace GC-MS instrument equipped with a DB-5 column (30 m × 0.25 mm; oil on DB-5 column under the same conditions. Identification of individual compounds was completed by the mass library, comparison of their mass spectra with the MS literature data (10)(11) and retention indices (RI). GC-FID peak area was used to calculate of the percentage compositions without the use of the correction factor. Molecular docking studies. The structure of compounds (1-4 and isoniazid), were drawn by Chem Draw Professional 15.0, and the structures were transferred to Chem3D and were saved as PDB format. Then in order to perform the energy minimization of the structures MM2 force field method was used. Finally minimized ligands in the PDB format were saved as PDBQT files using the Auto-Duck tool. The crystal structure of InhA with PDB ID: 1BVR was obtained from the RCSB Protein Data Bank (PDB). The protein structure was prepared by removing water and adding hydrogens and the protein file was saved as the PDBQT format for molecular docking studies.
Molecular docking was performed for determining binding modes of the ligands using Auto-Dock Vina software (12). The binding site residues of InhA with  (13). Binding site residues and grid box of InhA were shown in Fig. 2. In order to show interactions between ligand and receptor Discovery Studio Visualizer v4.5 was used.
Bacterial strains. An isolate of MDR M. tuberculosis was obtained from the microbial collection of Department of Medical Microbiology, Tehran University of Medical Sciences. The antimicrobial susceptibility testing was done according to CDC standard proportional method for multi-drug resistance confirmation of M. tuberculosis (14)(15). M. tuberculosis H37Rv was used as standard strain.
Determination of MIC. The serial dilution of each essential oil was completed in a concentration ranged from 0.250 to 1008 µg/mL in the sterile 96 wells. Also, Mueller-Hinton broth medium supplemented by 0.5% tween 80 was used as co-solvent as well as trace value. The suspensions were diluted in seven serial dilutions. The 100 μl of solution was transferred from column 1 to next column, and identical serial 1:2 dilutions were continued through column 7 (16). Each well was inoculated with 5μl of 0.5 McFarland standard turbidity of bacterial suspensions (17). A column without essential oil was inoculated as a growth control. A well with 80 μl of supplemented Middlebrook 7H9 medium and 20 μl of DMSO alone was also inoculated in each row for refuse anti-mycobacterial effect of DMSO. The plate was incubated at 37°C for 4 weeks. The wells were evaluated after 7, 14, 21 and 28 days and they were (Z)-ligustilide (6.22%), (Fig. 1).
Due to necessary treatment of TB infection, L. officinale essential oil was tested against MDR-TB. Results showed that L. officinale essential oil was relatively active on MDR-TB with MIC = 252 μg/ml. In this situation, MIC of isoniazid as pure compound and drug standard was equal to 4 μg/ml. It is notable that, the standard breakpoint value of isoniazid against MTB is equal to 2 μg/ml. α-Terpinenyl acetate (1), neocnidilide (2), βphellandrene (3) and (Z) -ligustilide (4) were chosen for molecular docking, because these compounds were specific and major compounds of L. officinale essential oil. Isoniazid as the first-line anti-tuberculous drug was selected as standard compound. Isoniazid has shown to act on M. tuberculosis by inhibiting a 2-trans-enoyl-acyl carrier protein reductase, called InhA (24). Due to this reason, InhA was selected for doing molecular docking. Docking score of compounds 1, 2, 3 and 4 was equal to -5.5, -6, -5.4, -6.4 kcal/mol respectively, that was comparable with docking score of isoniazid, by -4.6 kcal/mol.
As shown in Fig. 2, compound 1 has alkyl and Pi-alkyl interaction with ALA191, ILE21, PHE149, MET199 and PRO193 of binding site residues of the receptor. There are Pi-sigma, Pi-Pi, alkyl and Pi-alkyl interactions between compound 4 and ILE 215, ALA157, LEU218, PHE149, MET199 and PRO139 as the active site of the receptor. Interactions of compound 2 with receptor include, alkyl and Pi-alkyl with ALA157, MET155, TYR158, ILE215 LEU215 Table 1. The chemical composition of the volatile oil from L. officinale compared with the control wells. MIC was defined as the lowest essential oils concentration that exhibited no growth through visual inspection (17).

No
Compounds RI Percentage Identification

RESULTS
The essential oil was obtained by hydro distillation method on aerial parts of L. officinale and the

DISCUSSION
In the previous study by Mirjalili et al., antibacterial activity of essential oils from L. officinale at different fruiting stages (immature, mature and ripened) of the plant has been studied against Bacillus subtilis, Staphylococcus epidermidis, S. aureus and Escherichia coli. The mature and ripened fruit oils showed activity with MIC values of 0.9-1.8 mg/ml against B. subtilis, S. epidermidis. Also the essential oils at these stages exhibited sensitivity against E. coli with MIC=7.2 mg/ml (7).
The composition of L. officinale essential oil has been investigated widely and about 190 compounds have been reported. The presence of phthalide compounds is a characteristic for L. officinale essential oil. The content of phthalide compounds depends on the geographic location and weather conditions, critical phtalide compounds are propylidenephthalide, (E,Z)-ligustilide, (Z,E)-3-butylidenephthalide and dihydro-butylidenephthalide. Also there are terpenoids compounds including αand β-phellandrenes, αand β-pinenes, α-terpinyl acetate and α-terpineol (18). The principle compounds which are present in the essential oil extracted from aerial parts of L. officinale were β-phellandrenes (42.5%) and α-terpineol (27.9%) that was reported by Rizi et al. (19). In another study, significant compounds in leaves oil of L. officinale were β-phellandrene, α-terpinyl acetate and (Z)-ligustilide, and their content values were equal to 11.3%, 55.8% and 17.0% respectively (20).
M. tuberculosis as an intracellular pathogen causes pandemic tuberculosis (TB) (21) leading to the death of about 3 million people per year worldwide. Thus, it is necessary to treat TB through the use of novel drugs, especially in the case of multi-drug-resistant tuberculosis strain. MDR-TB is resistant to conventional drugs such as isoniazid and rifampicin (22)(23). Anti-TB activity of L. officinale oil is comparable to other results, for example the essential oil of laurel and anise had activity against M. tuberclosis with MIC = 100 μg/ml (3). Also the MICs related to three essential oils extracted from Salvia aratocensis, Turnera diffusa and Lippia Americana were equal to 125 μg/ml against M. tuberclosis (4).
α-Terpinenyl acetate, (Z)ligustilide and neocnidilide as specific and major compounds of L. officinale essential oil have more affinity to receptor even more than isoniazid, but isoniazid, unlike these compounds has common hydrogen bond. A conventional hydrogen bond is considered as an essential and vital interaction between drug and receptor. Due to this reason, it is fundamental to make change in the structure of compounds (1, 2 and 3) and investigate SAR and QSAR of these compounds to improve their anti-TB activity.

CONCLUSION
The L. officinale essential oil extracted form aerial parts of this plant was relatively active against MDR M. tuberculosis. It will be interesting to isolate compounds of the essential oil and evaluate their anti-TB activity separately, since based on the molecular docking studies done in the present study it has been shown that predominant compounds of the essential oil have had high affinity to inhibit the 2-trans-enoylacyl carrier protein reductase (InhA) as an important enzyme in M. tuberculosis cell wall biosynthesis.